This invention relates to nucleotide therapeutics, transplantation and immunology. More specifically, it relates to a newly discovered class of polynucleotide decoy molecules and methods for making cells and organs that are less likely to be rejected when transplanted into a recipient host using these polynucleotide decoys, which are capable of binding to a specific gene regulatory factor and reducing the expression of MHC class II transplantation antigens.
Among gene products that relate to transplantation antigens are the products of the Human Leukocyte Antigen (HLA) complex, also known as the major histocompatibility complex (MHC), located on the short arm of chromosome 6. The HLA antigens are divided into two classes depending on their structure. The genetic loci denoted HLA-A -B, and -C code for the HLA Class I antigens, and HLA-DP, -DQ and -DR code for the HLA Class II antigens.
HLA Class II molecules are composed of two non-covalently linked glycoproteins, the xcex1 chain and the highly polymorphic xcex2 chain. Each chain contains one extracellular domain, a transmembrane segment and a cytoplasmic tail. The structure of the xcex1 and xcex2 chains and their genes have been elucidated. All known Class II genes are similar in structure and encoded by exons 1-4, with exon 5 coding for an untranslated region. The DP, DQ and DR loci all consist of multiple genes. A total of twelve class II genes have been identified. In some haplotypes, some class II genes do not code for a functional peptide and are classified as pseudogenes.
Regulation of HLA class II antigen expression occurs in part through a series of promoter regions such as the J, W, X (including X1 and X2), and Y boxes, and the gamma interferon response element. The X (including X1 and X2) and Y boxes are known to be required in the transcriptional regulation of all class II promoters. Ono, S. J. et al., Proc. Natl. Acad. Sci. (USA) (1991) 88:4304-4308.
Transcription of HLA-DRxcex1 Class II can be activated by RF-X (Regulatory Factors-X) which binds to the X-box region (xe2x88x92110 to xe2x88x9295) of the DRxcex1 promoter. RF-X and its binding site, the X-box are unique and have a high specificity for each other. The DNA binding domain of RF-X consists of 91 amino acids with a basic stretch and shares no notable homology with other known DNA binding motifs (Reith et al. (1990), Genes Dev, Vol. 4(9), pp. 1528-40). RF-X binds only the X-box; substitutions in the X-box are generally not well tolerated by RF-X. (Hasegawa et al. (1991), Nucleic Acids Res., Vol 19(6), pp. 1243-49). The X-box sequence is an atypical promoter site, being neither palindromic nor dyad symmetric. Additionally no other sequence (using the program Eugene) shows exact homology with the X-box. The X-box is conserved in humans. No other known cloned transcription factors bind to this entire region of the X-box in the same manner.
HLA antigens are implicated in the survival of cell grafts or transplants. Although there is acceptable graft survival in the first year for nearly all types of transplants, by five and ten years after transplantation only 40-50% of all grafts are still functioning. This low rate is due to the relentless attack of the immune system on the graft. In addition, death rates of 1-5% are recorded even at the best transplant centers. Drugs are commonly used to control immune responses and prevent graft rejection, and death is often an indirect result of this drug administration.
The drugs used to control immune responses usually cause a non-specific depression of the immune system. A patient with a depressed immune system is far more susceptible to develop life-threatening infections and a variety of neoplasia. The low rate of long term success, and serious risks of infection and cancer are the two main challenges now facing the entire field of tissue and organ transplantation.
It has been suggested that graft rejection can be prevented or reduced by reducing the levels of exposed HLA antigens on the surface of transplant cells. Faustman, D. et al., Science (1991) 252:1700-1702, observed that xenograft survival was increased by masking HLA class I surface antigens with F(abxe2x80x2)2 antibody fragments to HLA class I or tissue specific epitopes.
One way to reduce the level of cell surface transplantation antigens is to retard (downregulate) the expression of the transplantation antigen genes.
Generally, eucaryotic gene expression may be regulated at any of the steps from DNA transcription to RNA translation to protein; and it is generally agreed that gene expression is at the level of transcription. In order for transcription to occur, transcription factors must bind distinct regulatory sites or promoters on the gene. Once bound, transcription factors may interact with RNA polymerase or other factors to activate or repress transcription. Some transcription factors are constitutively expressed in specific cells while others may be transiently activated in response to various physiological signals (such as cAMP, IFN-xcex3, etc.). Thus in a given cell transcription of particular genes depends on which transcription factors are present in that cell type and/or whether the signals to activate the transcription factors are present.
Agents such as actinomycin (an intercalator) have been used to block transcription in a nonspecific manner. A variety of approaches to sequence-specific gene modulation include use of antisense oligonucleotides and antigene oligonucleotides (triple helix formers). These are limited in general or in particular instances. Antisense oligonucleotides block gene expression by targeting mRNA while triple helix forming oligos target double-stranded DNA. Inaccessibility of the target mRNA due to RNA secondary structure can limit the usefulness of antisense methods; triple helix approaches are limited by poor nuclear access, chromatin structure (bypassing histones), and the need for targeting a homopurine-homopyrimidine stretch. Degradation of oligonucleotides by exonucleases an excessive binding to untargeted cellular factors can limit the effectiveness of both antisense and triple helix methods. Chemical modification of oligos can improve nuclease resistance but can also result in increased toxicity, reduced binding affinity, and lower activity (Cook 1991, Crooke 1991).
J. T. Holt (1991), Antisense Res. Dev., Vol. 1(4), pp. 65-9, has shown that by providing excess DNA binding sites, specific transcription factors can be quenched and are thereby prevented from binding to endogenous DNA.
Chu et al. (1991) Nucl. Acids Res. 19:6958, describe DNA structures in  hairpin  and  dumbbell  configurations containing CRE and TRE sequences, and reported using them in vitro as substitutes for regular double-stranded DNA to bind CREB and JUN, respectively, in gel shift assays.
Chu et al. (1992) Nucl. Acids Res. 2:5857-5858, demonstrate that in nuclear extracts, dumbbell DNA is much more stable than double-stranded or hairpin DNA. However, the nicking of dumbbell DNA in human serum, caused by endonuclease degradation of the single-stranded loops, is a potential problem, since it converts dumbbell DNA to a double-stranded form that is no more stable in the nucleus than standard double-stranded DNA molecules.
International patent publication WO92/19732 describes  closed  oligonucleotides that can be used as  sense  or  antisense  molecules, with the advantage of being resistant to exonucleases. Among the variety of  closed  structures are  dumbbell  configurations in which the ends are closed by virtue of addition links of thymidine nucleotides. Use of the closed  sense  oligonucleotides to bind protein factors having as affinity for RNA or DNA sequences or structures is suggested.
Single-strand circular DNA where a portion becomes double-stranded have been used as an experimental system for studying local thermal stability in DNA (Wemmer et al. (1985), Nucl. Acid. Res., Vol. 13(23), pp. 8611-21); as models for hairpins and cruciforms (Erie et al. (1989), Biochemistry, Vol. 28(1), pp. 268-73); and as models for comparison to nicked or gapped DNA (Snowden-Ifft et al. (1990), Biochemistry, Vol. 29(25), pp. 6017-25).
We have discovered a class of polynucleotide decoys that competitively bind transcription factors necessary for transcription of MHC-II genes. Particularly, for example, we have identified oligonucleotides that mimic the X-Box of MHC-II and that competitively bind the MHC-II transcription factor RF-X. Exposing the cell to such an oligonucleotide can result in production of a MHC-II-depleted cell. That is, the polynucleotide decoys of the invention act as modulators of MHC-II expression. The invention can be used, therefore, to inhibit the expression of HLA molecules on the surface of donor organ/cell, in order to reduce its visibility to the host""s immune system.
The polynucleotide decoy of the invention is a centrally double-stranded (but potentially lacking as many as two base pairings),  dumbbell  shaped structure, with linking cap oligonucleotides that is covalently closed in some embodiments and in other embodiments is linear. The single-stranded caps linking each of the two ends of the polynucleotide decoy are present to stabilize the structure, prevent degradation by exonucleases, and are not intended to be involved in recognition of the protein of interest. Compared to antisense and antigene approaches, target inaccessibility may be of less concern for the polynucleotide decoy, because its target is a protein which may be found in the cytoplasm as well as the nucleus. Lower doses of the polynucleotide decoy may suffice to effectively block transcription of the gene of interest, as compared for example to dosages of nucleotides required for an antisense approach, because transcription factors are ordinarily present in low copy number.
Preferred polynucleotide decoys are short covalently closed nucleotides, with an entirely double-stranded internal segment, and wherein the polynucleotide decoys have a sequence that mimics the double-stranded sequence of the X-box, with polythymidine segments linking the ends together. Administration of a polynucleotide of the invention results in downregulation of HLA II DRxcex1 proteins or mRNA. The polynucleotide decoys of the invention can be useful in regulating gene expression in the HLA system and in other systems as well.
The invention contemplates the development of a  universal donor cell  reduced in one or more HLA antigens by treatment of the cell with a polynucleotide decoy of the invention. The absence of certain HLA antigens on the surface of donor cells, tissues or organs comprising these cells will cause them not to be recognized as foreign and not to elicit a rejection response. By the selective introduction of polynucleotide decoys into a cell it is possible to block the expression of MHC class II genes, particularly constitutive expression, thereby rendering a graft  invisible  to the immune system. Thus, the problem of rejection is eliminated without nonspecific suppression of the immune system, and the immune system remains active to defend against infection and neoplasia. By implementing the methods of the present invention, even xenogeneic donor cells, tissues and organs can be rendered  invisible  to the immune system. Thus, the target cells or organs of the present invention may be derived from non-human mammalian species, made into transplantation antigen-depleted cells or universal donor organs, respectively, by a method of the invention, and subsequently transplanted into a human individual.
In particular the present invention embodies a class of polynucleotide decoys which comprise: (a) an internal oligonucleotide (I) having a length of X bases, where X is a number from about 10 to about 40; (b) two cap oligonucleotides (P1 and P2), each having a length of from about 3 to about 8 bases, wherein each of said cap oligonucleotides is comprised of bases which are unable to bind to any other base within the same cap oligonucleotide; (c) a first complementary oligonucleotide (C1) having a length of Q bases, where Q is a number from about 5 to (Xxe2x88x925), said C1 having a 3xe2x80x2 to 5xe2x80x2 nucleic acid sequence capable of Watson-Crick-type binding to the first Q bases in the 5xe2x80x2 to 3xe2x80x2 nucleic acid sequence of said I; and (d) a second complementary oligonucleotide (C2) having a length of Z bases, where Z is a number greater than equal to about 5 and from ((Xxe2x88x92(Q+about X/8)) to (Xxe2x88x92Q), said C2 having a 5xe2x80x2 to 3xe2x80x2 nucleic acid sequence capable of Watson-Crick-type binding to the first Z bases in the 3xe2x80x2 to 5xe2x80x2 nucleic acid sequence of said I; wherein the 3xe2x80x2 end of said C1 is covalently linked to the 5xe2x80x2 end of said P1, the 3xe2x80x2 end of said P1 is covalently linked to the 5xe2x80x2 end of said I, the 3xe2x80x2 end of said I is covalently linked to the 5xe2x80x2 end of said P2, and the 3xe2x80x2 end of said P2 is covalently linked to the 5xe2x80x2 end of said C2; wherein at least one of the oligonucleotides selected from the group consisting of said C1, said C2, and said I, comprises an RF-X recognition sequence of Type-1 or Type-2; and wherein said polynucleotide decoy is capable of binding to an RF-X transcription factor.
The present invention also embodies methods of making the polynucleotide decoys of the invention comprising covalently linking oligonucleotides such that the resulting product has the structure of a polynucleotide decoy of the invention.
The present invention embodies methods for making an MHC-II-depleted cell from a target cell, the method comprising: (a) obtaining the target cell; and (b) exposing the target cell to a polynucleotide decoy of the invention, said polynucleotide decoy being preset in an amount sufficient to make the target cell a MHC-II-depleted cell. The present invention additionally embodies the MHC-II-depleted cells prepared by the fore-mentioned method.
The present invention further embodies polynucleotide decoys of the invention for use in the preparation of a composition for treating target cells to make them MHC-II-depleted cells.
The present invention further embodies MHC-II-depleted donor organs prepared by the method comprising: (a) obtaining a target organ from an individual; and (b) exposing the target organ to a polynucleotide decoy of the invention, said polynucleotide decoy being present in an amount sufficient to make the target organ a MHC-II-depleted donor organ.
The present invention further embodies methods of treating an individual with an autoimmune disease characterized by dysfunctional expression of an MHC class II antigen, the method comprising administering to that individual a polynucleotide decoy of the invention in an amount sufficient to inhibit expression of the MHC class II antigen.
The present invention also embodies methods of treating an individual infected with hepatitis B virus, the method comprising administering to that individual a polynucleotide decoy of the invention, in an amount sufficient to inhibit expression of hepatitis B surface antigen.
The present invention further embodies methods of treating an individual infected with cytomegalovirus, the method comprising administering to that individual a polynucleotide decoy of the invention in an amount sufficient to inhibit RF-X binding to the enhanced factor C sites of the cytomegalovirus genome.
The present invention further embodies methods of using a polynucleotide decoy to obtain a substantially purified, covalently closed polynucleotide decoy, comprising the steps of: (a) providing a linear polynucleotide decoy of the invention; (b) treating said linear polynucleotide decoy with a kinase enzyme, thereby obtaining 5xe2x80x2 phosphorylated polynucleotide decoy; (c) heating said 5xe2x80x2 phosphorylated polynucleotide decoy to inactivate said kinase enzyme, thereby obtaining heated, 5xe2x80x2 phosphorylated polynucleotide decoy; (d) cooling said heated, 5xe2x80x2 phosphorylated polynucleotide decoy mixture slowly to facilitate intramolecular annealing, thereby obtaining cooled, 5xe2x80x2 phosphorylated polynucleotide decoy; (e) treating said cooled, 5xe2x80x2 phosphorylated polynucleotide decoy with a ligating enzyme, thereby obtaining a covalently closed polynucleotide decoy; and (f) purifying said covalently closed polynucleotide decoy, thereby obtaining a substantially purified, covalently closed polynucleotide decoy.
The present invention also embodies nucleic acids comprising a linear polynucleotide decoy of the invention.
The present invention further embodies host cells comprising a nucleic acid comprising a linear polynucleotide decoy of the invention.